Heterologous expression and kinetic characterization of the α, β and αβ blend of the PPi-dependent phosphofructokinase from Citrus sinensis
Introduction
Pyrophosphate dependent phosphofructokinase (PPi-PFK, EC 2.7.1.90) is a glycolytic/gluconeogenic enzyme that phosphorylates fructose-6-phosphate (Fru6P) to fructose-1,6-bisphosphate (Fru1,6bisP) using inorganic pyrophosphate (PPi) as a phosphoryl donor in a reversible enzymatic reaction [[1], [2], [3]].Fru6P + PPi ↔ Fru1,6bisP + Pi
The enzyme was first discovered in 1974 in Entamoeba histolytica [3] (a human pathogenic protozoan). It was later found in Gram-positive bacteria [1,4] and also in higher plants and algae [5,6]. The occurrence of PPi-PFK in these organisms provides an alternative to the cytosolic ATP-dependent phosphofructokinase (ATP-PFK, EC 2.7.1.11) that also phosphorylates Fru6P to Fru1,6bisP during glycolysis, but using ATP instead of PPi. After many years of research, it is clear that the major regulation point in non-plant glycolysis is exerted at the level of ATP-PFK, which controls the conversion of Fru6P into Fru1,6bisP [[7], [8], [9]]. But in plants, glycolysis is mainly regulated at the level of phosphoenolpyruvate metabolism, being the phosphorylation of Fru6P a secondary control point [8].
The kinetic, regulatory and structural characterization of PPi-PFK from plants was mainly achieved by purifying and characterizing the enzyme from different plant species and tissues [[10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23]]. These studies established that PPi-PFK in plants is generally composed of α and β subunits arranged in different hetero-oligomeric structures. Brassica nigra and Musa cavendishii have a PPi-PFK with a heterooctameric structure (α4β4) [21,23], while the enzyme from Daucus carota showed a heterotetrameric (α2β2) conformation [24]. Potato tuber PPi-PFK has been structurally described either as a symmetrical α2β2 tetramer [16] or as a α4β4 heterooctamer [19]. Additionally, in Ananas comosus the active enzyme is a dimer [22]. Regulation of the enzyme is exerted primarily by activation by Fru2,6bisP [8,9,20,22]. Based on 3D modelling of grapefruit PPi-PFK and sequence data, several authors [[25], [26], [27]] have suggested that only the β subunit carries the catalytic site, and that the α subunit is essential for activation as the Fru2,6bisP docking site is actually formed in the interface of both subunits [25,26]. In Ricinus communis cotyledons, the profile of PPi-PFK activity during germination closely follows the level of β subunit present [28]. However, the existence of enzyme forms activated by Fru2,6bisP but lacking the α subunit [22] makes things not clear in this subject. Phospoenolpyruvate is an allosteric inhibitor of the enzyme, and also has the effect of decreasing Fru2,6bisP levels [8]. The relative amount of α and β subunits in different plant tissues depends on factors like nutrient availability, environmental stress or developmental stage of the plant tissue [19,21,29]. It has been suggested that a coarse regulation of the enzyme activity would be exerted by such a differential subunit expression [28,29]. For this and other reasons the role of PPi-PFK in relation to stress response and its function in plant growth has been investigated. Lim et al. (2009) [30] showed that Arabidopsis plants with altered PPi-PFK expression (overexpressing or repressing the enzyme) have altered growth patterns, in a manner that high PPi-PFK levels promote earlier development, while decreased expression retards it, in contest to earlier findings showing the opposite (i.e. no effect on growth) in genetically altered potato [31], tobacco [32] and sugarcane plants [15]. Moreover, they showed that these altered expression patterns affect the expression levels of several metabolically important enzymes, which suggests that PPi-PFK has a widespread effect on general metabolism that goes beyond its activity. In a later paper Lim et al. [33], the same group obtained double and quadruple mutants of Arabidopsis deficient in both α1 or α2, β1 or β2 subunits or in all them. They found that Pi or N deprivation did not affect significantly the growth rate compared to controls. However, these authors observed that PPi-PFK is necessary to withstand salt and osmotic stress at germination and during seedling growth and concluded that the enzyme is needed during acclimation to these type of stress conditions, perhaps because of PPi-PFK´s central role in carbohydrate metabolism, that leads to an accumulation of compatible solutes during salt/water stress. Nielsen and Stitt [32] observed that the base tip of young leaves, an actively growing tissue, contains higher PPi-PFK activity, supporting the hypothesis that the enzyme´s roles in young and mature tissue are different. These authors, as others, also noted that transformants with reduced PPi-PFK (i.e. less than 10% of the wild type) did not show a corresponding metabolic shift. Instead, higher levels of Fru2,6bisP could compensate for the activity loss. Notwithstanding the extensive research dedicated to PPi-PFK, an in-depth examination of the enzyme structure-to-function relationship is still lacking and many questions remain open. Perhaps the main causes are the technical aspects regarding the isolation of the isolated subunits from the plant tissues.
In this work, two pfp genes from Citrus sinensis (orange) coding for α and β subunits of PPi-PFK were cloned and expressed by a recombinant strategy. Using Escherichia coli as a host, the individual expression of the recombinant α and β subunits and also the coexpression of both was achieved. All the purified proteins were studied to determine its kinetic parameters and comparatively analyzed in order to better understand the role of each subunit of the plant enzyme.
Section snippets
Chemicals
Protein standards, antibiotics, isopropyl-thiogalactoside (IPTG), Fru6P, Fru2,6bisP, PPi, Pi, and oligonucleotides were obtained from Sigma-Aldrich. All other reagents were of the highest quality available.
Gene amplification
The pfpα and pfpβ genes (respectively coding for subunits α and β of PPi-PFK) from C. sinensis (sweet orange, Valencia Late variety) were amplified by PCR by using orange endocarp mRNA, prepared by standard protocols [34] and accurate primer oligonucleotides. Since at the moment of these
Expression and purification of the α, β and αβ PPi-PFK from orange
The sequence of the genes from C. sinensis coding for α (Csipfpα, NCBI EU302909.1) and β (Csipfpβ, NCBI EU302910.1) PPi-PFK subunits were amplified by PCR with specific oligonucleotides and the plant cDNA. The strategies for heterologous expression of the different enzyme conformations are summarized in Fig. 1. The scheme shows that the individual expression of His-Tagged α subunit was achieved in E. coli BL21 Rosetta™ (DE3) cells transformed with [pET28c/Csipfpα] construction, while the β
Acknowledgments
This work was supported by ANPCyT (PICT 2015-1074 to FEP, PICT 2016-0091 to KEJT, PICT 2014-3256 and 2015-1767 to AAI, PICT 2014-2264 to CVP), and Universidad Nacional del Litoral (CAID 2016 to AAI). RJM is doctoral fellow and EM, FEP, KEJT and AAI are Researchers from CONICET. CVP is at present a Postdoctoral fellow from Institut Pasteur de Montevideo, Uruguay.
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Both authors contributed equally to the work.